Figure 1.
Subcellular localization of EG-porphyrin derivatives.
4T1 cells were loaded with EG-porphyrin derivatives (left panels) and specific probes for endoplasmic reticulum (ER) and lysosomes (middle panels). An overlay of EG-porphyrin derivatives fluorescence with ER-Tracker or LysoTracker fluorescence is shown in the right panels. Scale bars: 10 µm.
Figure 2.
Effect of kinase inhibitors on EG-porphyrin-mediated phototoxicity in 4T1 and HL60 cells.
4T1 cells (A) or HL60 cells (B) were loaded with EG-porphyrin derivatives for 16 h, washed and then preincubated for 1 h JNKII inhibitor (5 µM) and PD169316 (10–20 µM) before light exposure. After irradiation the cells were further cultivated and cell viability was determined 24 h post-PDT by the trypan blue exclusion method. Cells treated with EG-porphyrin derivatives without irradiation were used as controls (dark). The percentage of dead cells was expressed as the mean ± SD (n = 4). ** P<0.01 represents statistical differences between PDT-treated cells vs. PDT-treated cells in the presence of JNKII or PD169316. For Western blot analysis cells were pre-incubated for 1 h with JNKII inhibitor (SP600125) or p38 inhibitor (PD169316) and 2 h post-irradiation analyzed with specific antibodies recognizing the phosphorylated forms of p38 and c-Jun by Western blot (P-p38 and P-c-Jun, respectively). Equal loading is demonstrated by total c-Jun and p38 protein. (C) Effect of p38 deletion on apoptotic cell death. Mouse embryo fibroblasts (MEFs) with intact (wt) and inactivated p38α−/− (KO) gene were exposed to EG-porphyrin-mediated PDT. Cell viability was evaluated 24 h post-PDT by the trypan blue exclusion method. **P<0.01 represents statistical differences between wt MEF vs. KO MEF exposed to PDT.
Figure 3.
Role of ROS generation in EG-porphyrin-mediated cell death.
(A) Effect ROS scavengers on cell viability after PDT. 4T1 cells were loaded with EG-porphyrin derivatives for 16 h, washed and then pre-incubated for 1 h with L-histidine (20 mM), trolox (5 mM), NAC (15 mM), tiron (10 mM), DMSO (1%), PEG-catalase (300 U/ml) and sodium pyruvate (Na Pyruv) (10 mM) before light exposure. Similarly, HL60 cells were pre-incubated with L-histidine (20 mM), trolox (4 mM), NAC (5 mM), tiron (10 mM), DMSO (1%), PEG-catalase (300 U/ml) and sodium pyruvate (Na Pyruv) (10 mM). After irradiation the cells were further cultivated and cell viability was determined 24 h post-PDT by the trypan blue exclusion method. Cells treated with EG-porphyrin derivatives without irradiation were used as controls (dark). The percentage of dead cells was expressed as the mean ± SD (n = 3). *P<0.050, ** P<0.01 represents statistical differences between PDT-treated cells vs. PDT-treated cells in the presence of L-histidine, trolox, NAC, tiron and DMSO. (B) ROS detection. 4T1 cells incubated with EG-porphyrin derivatives were washed and loaded with 10 µM APF(3′-(p-aminophenyl) fluorescein) in the presence or absence of L-histidine (20 mM) or DMSO (1%) for 30 min. Dye-loaded cells were then stimulated with UV light for 10 s under a DM IRB microscope (Leica) and fluorescence images were acquired immediately using a ×10 objective. (C) In situ detection of ROS generation. 4T1 cells grown on coverslips were sequentially loaded with porphyrin derivatives and APF probe. After 30 min incubation with the APF probe cells were exposed to UV light for 10 s and fluorescence images were acquired immediately using a ×63 objective. Fluorescent images of EG-porphyrin derivatives (left panels), APF probe (middle panels) and their overlay (right panels). Scale bars: 10 µm.
Figure 4.
The role of Ca2+ in photoinduced apoptosis by EG-porphyrin derivatives.
(A) Intracellular calcium levels in HL60 and 4T1 cells. The effect EG-porphyrin derivatives on cytosolic Ca2+ levels was monitored by flow cytometry of laser-illuminated cells containing fluorescent Ca2+ indicator Fluo-4-AM (4 µM) (Molecular Probes). Cells treated in the same way but without porphyrin were used as controls. (B) Effect of BAPTA and L-histidine on the intracellular Ca2+ level. HL60 cells were loaded with EG-porphyrin derivatives for 16 h, washed and then preincubated for 1 h with ROS scavenger L-histidine (20 mM) or 2 h with calcium chelator BAPTA-AM (5 µM). The cytosolic Ca2+ level was monitored by flow cytometry using fluorescent Ca2+ indicator Fluo-4-AM (4 µM). (C) Effect of BAPTA-AM preincubation on photoinduced apoptosis. Cells loaded with EG-porphyrin derivatives were pre-incubated with the membrane-permeable intracellular Ca2+ chelator BAPTA-AM (5 µM HL60, 10 µM 4T1) for 2 h to inactivate released Ca2+ and then irradiated with 2.5 Jcm−2 of light at 500±20 nm. This corresponds to an LD70 PDT dose. Cell viability was determined 24 h post-photodynamic therapy by the trypan blue exclusion method. The percentage of dead cells was expressed as mean ± SD (n = 4). **P<0.01, ***P<0.001 represents statistical differences between PDT-treated cells vs. PDT-treated cells in the presence of BAPTA-AM.
Figure 5.
(A) Cells loaded with EG-porphyrin derivatives were pre-treated with Ru360 (5 µM 4T1, 4 µM HL60) for 1 h to inhibit Ca2+ mitochondrial uptake. After irradiation the cells were further incubated in medium and their viability was determined 24 h later by the trypan blue exclusion method. The percentage of dead cells was expressed as the mean ± SD (n = 3–6). **P<0.01, ***P<0.001 represents statistical differences between PDT-treated cells vs. PDT-treated cells in the presence of Ru360. (B) Cells were preincubated for 1 h with Ru360 (4–5 µM) and 2 h post-irradiation the cytosolic, mitochondrial and nuclear extracts were prepared and analyzed by Western blotting with specific antibody recognizing cytochrome c, GAPDH and PARP. Results are representatives of at least three experiments.
Figure 6.
Effect of EG-porphyrin derivatives on Ca2+ signaling pathway.
(A) Western blot analysis of fodrin, caspase-12 (4T1 cells) and caspase-4 (HL60 cells). Cells treated with EG-porphyrin derivatives were harvested at various times after irradiation and subjected to Western blot analysis with antibodies recognizing fodrin and caspase-12. Reprobing with β-actin antibody was used to confirm equal loading. The arrowheads point to activated forms. (B) Pretreatment of 4T1 cells with BAPTA-AM (10 µM) resulted in the inhibition of fodrin, caspase-9 and caspase-3 activation caused by mTPP(EG)4-PDT. 4T1 cells loaded with porphyrin derivatives were pre-incubated for 2 h with BAPTA-AM and then exposed to light (2.5 Jcm−2). At various times after irradiation the cells were lysed and analyzed with antibody recognizing fodrin, full-length and cleaved p39 form of caspase-9 and full-length and cleaved p17 form of caspase-3 on Western blots. (C) Effect of calpain inhibitor PD150606 on viability of 4T1 and HL60 cells. Cells were incubated with PD150606 (20 µM) for 1 h and then irradiated. Cell viability was estimated after 24 h by the trypan blue exclusion method. The percentage of dead cells was expressed as the mean ± SD (n = 4–5). **P<0.01, ***P<0.001 represents statistical differences between PDT-treated cells vs. PDT-treated cells in the presence of PD150606. (D) The effect of calpain inhibitor, ROS scavengers (L-histidine and trolox), and caspase inhibitor (Z-VAD-FMK) on the activation of fodrin. Cells were treated as described in Materials and Methods, harvested 1 h post PDT treatment and analyzed by Western blot. Equal protein loading is demonstrated by actin reprobing. The viability of cells subjected to simultaneous treatment done in parallel is presented under the Western panel.
Figure 7.
Effect of Capn4 siRNA on the demise of PDT-treated cells.
(A) Western blot analysis of capn4/capns1 and fodrin in 4T1 cells analyzed on the 3rd day after Capn4 siRNA transfection. Transfected cells were subjected to EG-porphyrin-mediated PDT, harvested and resolved by Western blot analysis. Equal protein loading is demonstrated by actin reprobing. (B) Viability of cells transfected with Capn4 siRNA. Transfected and control cells were subjected to EG-porphyrin-mediated PDT and cell viability was monitored 24 h later. The percentage of dead cells was expressed as the mean ± SD (n = 3). *P<0.05 represents the statistical difference between transfected PDT-treated cells vs. non-transfected or control siRNA-transfected PDT-treated cells.
Figure 8.
Kinetics of PERK pathway activation by EG-porphyrin-mediated PDT.
(A) The induction of ER stress proteins (P-PERK, P-eIF2α, ATF4, CHOP) detected by Western blot analysis. The activity of PERK and eIF2α was determined by phospho-specific antibodies (p-) and then the membranes were reprobed with antibodies to total PERK and eIF2α. Equal protein loading is demonstrated by actin reprobing. As a positive control were used cells treated with 1 µM thapsigargin (TG) for 6 h. (B) qRT-PCR analysis of total mRNA isolated from PDT-treated 4T1 and HL60 cells. cDNA was prepared from total mRNA and quantitative real-time RT-PCR for ATF4, CHOP and β-actin mRNA was performed. mRNA fold induction values were calculated from ATF4/actin and CHOP/actin ratios as described in Materials and Methods. Experiments were performed in triplicate.
Figure 9.
Effect of PERK siRNA on the demise of PDT-treated cells.
PERK deficiency protects against mPTT(EG)4-mediated cell death. (A) Western blot analysis of PERK, ATF4 and CHOP in 4T1 cells after PERK siRNA transfection. Transfected cells were subjected to EG-porphyrin-mediated PDT, harvested and resolved by Western blot analysis. Equal protein loading is demonstrated by actin reprobing. As a positive control were used cells treated with 1 µM thapsigargin (TG) for 6 h. (B) Viability of cells transfected with PERK siRNA. Transfected and control cells were subjected to EG-porphyrin-mediated PDT and cell viability was monitored 24 h later. The percentage of dead cells was expressed as the mean ± SD (n = 7–14). **P<0.01 represents the statistical difference between transfected PDT-treated cells vs. non-transfected or control siRNA-transfected PDT-treated cells. (C) Immunostaining of CHOP after EG-porphyrin derivative-mediated PDT in PERK knockdown cells. Cells were fixed 2 h post PDT, immunostained and observed with a fluorescence microscope. The left panel includes untransfected cells, the middle panel cells transfected with control siRNA and the right panel cells transfected with PERK siRNA. (D) Cell viability of MEF-wt and MEF-KO evaluated 24 h post-EG-porphyrin-PDT (350 nM mTPP(EG)4, 3.5 µM pTPP(EG)4, 700 nM pTPPF(EG)4). Verification of the PERK−/− phenotype in MEFs-KO cells by Western blot. The percentage of apoptotic cells was expressed as the mean ± SD (n = 6). ***P<0.001 represents statistical differences between PDT-treated MEF-wt cells vs. PDT-treated MEF-KO cells. (E) Fluorescence microscopy demonstrates similar cellular uptake of EG-porphyrin derivatives in MEFs-wt and MEF-KO [800 nM mTPP(EG)4, 5 µM pTPP(EG)4, and 2 µM pTPPF(EG)4].